Adult Prg4+ progenitors repair long-term articular cartilage wounds in vivo

The identity and origin of the stem/progenitor cells for adult joint cartilage repair remain unknown, impeding therapeutic development. Simulating the common therapeutic modality for cartilage repair in humans, i.e., full-thickness microfracture joint surgery, we combined the mouse full-thickness injury model with lineage tracing and identified a distinct skeletal progenitor cell type enabling long-term (beyond 7 days after injury) articular cartilage repair in vivo. Deriving from a population with active Prg4 expression in adulthood while lacking aggrecan expression, these progenitors proliferate, differentiate to express aggrecan and type II collagen, and predominate in long-term articular cartilage wounds, where they represent the principal repair progenitors in situ under native repair conditions without cellular transplantation. They originate outside the adult bone marrow or superficial zone articular cartilage. These findings have implications for skeletal biology and regenerative medicine for joint injury repair.

B-C. mouse knee surgical wounds at time 0; (B), using needle; (C), using core biopsy punch.Arrow points to the surgical wound.Scale Bar: 1 mm.Individual panels might be adjusted for brightness and contrast and displayed using Fiji.The original images may be cropped to display the articular cartilage for adequate visualization.Arrow: superficial soft tissue surrounding the wound proper.
Long Arrows: site of subchondral bone fracture.
Patella tendon was cut during tissue processing to ensure penetration of tissue processing reagents.
Individual panels might be adjusted for brightness and contrast and displayed using Fiji.The original images may be cropped to display the articular cartilage for adequate visualization.Red: tdTomato.Blue: DAPI nuclei stain.
Individual panels might be adjusted for brightness and contrast and displayed using Fiji.The original images may be cropped to display the articular cartilage for adequate visualization.Individual panels might be adjusted for brightness and contrast and displayed using Fiji.The original images may be cropped to display the articular cartilage for adequate visualization.B-E.21 dpi: Alcian blue staining of Prg4 creERt;tdTom knee wound of a nearby section to that shown in figure 3A, given 4mg tamoxifen, followed by washout, injury and euthanasia at 21 dpi.Scale Bar: 500 µm (B), 50 µm elsewhere.
Individual panels might be adjusted for brightness and contrast and displayed using Fiji.The original images may be cropped to display the articular cartilage for adequate visualization.

B
Injury Alcian Blue staining of a nearby section to that shown in figure1G(given tamoxifen 8mg and using punch).
images of Prg4 creERt;rosa tdTomato mouse knee following similar protocol as in figure 2A (homeostasis), given higher tamoxifen at 12mg.Alcian Blue stained nearby section to figure2I, with mice injured using needle and euthanized at 60 dpi.Dotted contours: the wound border.Scale Bar: 500 µm (A), 50 µm (B).Individual panels might be adjusted for brightness and contrast and displayed using Fiji.The original images may be cropped to display the articular cartilage for adequate visualization.Alcian blue (A,B) and fluorescent confocal (C) images of Prg4 creERt; rosa tdTomato knee wound (using punch), receiving only corn oil and euthanized at 60 dpi (n=2).Dotted contours: the wound border.Scale Bar: 500 µm (A), 50 µm (B).
Aggrecan mRNA in situ hybridization negative controls using negative control mRNA probes and anti-RFP antibody.(A): overview.(B,C): high magnification insets.Dotted contours: the wound border.Blue: DAPI.Red: tdTomato.Red was pseudo-colored based on the staining signals of anti-tdTomato secondary antibody.Green: ACD negative control mRNA probe.Green was pseudo-colored based on the staining results of fluorescent mRNA in situ hybridization using far-red fluorophores to eliminate background associated with cartilage matrix auto-fluorescence.Col2 mRNA in situ hybridization negative controls, as in A-C.Scale bars: 50 µm.Individual panels might be adjusted for brightness and contrast and displayed using Fiji.The original images may be cropped for adequate visualization.Dotted contours: the wound border.Blue: DAPI.Red: tdTomato.Red was pseudo-colored based on the staining signals of anti-tdTomato secondary antibody.Green: ACD negative control mRNA probe.Green was pseudo-colored based on the staining results of fluorescent mRNA in situ hybridization using far-red fluorophores to eliminate background associated with cartilage matrix auto-fluorescence.images of Prg4 CreERt; rosa tdTomato with injury first then tamoxifen (reversing the sequence of tamoxifen and injury used in figure 2), to detect Prg4 expressing cells in post-injury marrow and wounds, showing post-injury marrow, wounds ("w"), articular chondrocytes, and synovium ("syn").Tamoxifen 4 mg was given for all time points, except 6mg for 4,6,8,11 dpi with euthanasia within 2-4 days.Dashed contours outline the superficial soft tissue surrounding the wound.Dotted contours: the wound border.At 20 dpi, soft tissue surrounding the wound contained some tdTomato+ cells but these were not included in wound cell counts due to their locations outside the wound proper.Red: tdTomato.Blue: DAPI nuclei stain.Scale bars: 50 µm.Individual panels might be adjusted for brightness and contrast and displayed using Fiji.The original images may be cropped to display the articular cartilage for adequate visualization.B. Quantitative summary: % Prg4 expressing (tdTomato) cells in tissues post-injury.(n=2 for groups given tamoxifen at 0, 16 or 18 dpi, and n=1 for the other groups).Two-way ANOVA: interaction, F=0.7812 (df between groups=12 and within groups=9), p=0.6622; time effect (wound dpi), F=0.7492 (df between groups=6 and within groups=9), p=0.6255; tissue effect (SFZ chondrocytes, marrow, and wound), F=150.2 (df between groups=2 and within groups=9), p=<0.0001;Tukey's multiple comparisons shown.All data were presented as mean ± s.d.